The protein kinase C (PKC) family comprises serine/threonine kinases involved in signal transduction pathways regulating cell proliferation and differentiation. Chronic activation of PKC results in abnormal cellular proliferation and tumor formation. Interest in PKC was stimulated by the finding that PKC is the major, and perhaps only, cellular receptor through which a class of tumor-promoting agents called phorbol esters exert their pleiotropic effects on cells (Gescher et al., Anti-Cancer Drug Design, 1989, 4, 93-105). Phorbols capable of tumor production can mimic the effect of diacylglycerol (DAG) in activating PKC, suggesting that these tumor promoters act through PKC and that activation of this enzyme is at least partially responsible for the resulting tumorigenesis (Parker et al., Science, 1986, 233, 853-866).
Increased tumorigenicity is also correlated with overexpression of PKC in cultured cells inoculated into nude mice. A mutant form of PKC induces highly malignant tumor cells with increased metastatic potential. Sphingosine and related inhibitors of PKC activity have been shown to inhibit tumor cell growth and radiation-induced transformation in vivo (Endo et al., Cancer Research, 1991, 51, 1613-1618); Borek et al., Proc. Natl. Acad. Sci., 1991, 88, 1953-1957). A number of experimental or clinically useful anti-cancer drugs show modulatory effects on PKC. Therefore, inhibitors of PKC may be important cancer-preventive or therapeutic agents. PKC has been suggested as a plausible target for more rational design of conventional anti-cancer drugs (Gescher, A. and Dale, I. L., Anti-Cancer Drug Design, 1989, 4, 93-105).
Experiments also indicate that PKC plays an important role in the pathophysiology of hyperproliferative skin disorders such as psoriasis and skin cancer. Psoriasis is characterized by inflammation, hyperproliferation of the epidermis and decreased differentiation of cells. Various studies indicate a role for PKC in causing these symptoms. PKC stimulation in cultured keratinocytes can be shown to cause hyperproliferation. Inflammation can be induced by phorbol esters and is regulated by PKC. DAG is implicated in the involvement of PKC in dermatological diseases, and is formed to an increased extent in psoriatic lesions.
Inhibitors of PKC have been shown to have both antiproliferative and antiinflammatory effects in vitro. Some antipsoriasis drugs, such as cyclosporine A and anthralin, have been shown to inhibit PKC. Inhibition of PKC has been suggested as a therapeutic approach to the treatment of psoriasis (Hegemann, L. and Mahrle, G., Pharmacology of the Skin, H. Mukhtar, ed., 1992, CRC Press, Boca Raton, Fla., p. 357-368).
PKC is not a single enzyme, but a family of enzymes. At the present time at least ten isoforms (isozymes) of PKC have been identified: the "conventional" isoforms .alpha., .beta., and .gamma., the "novel" isoforms .delta., .epsilon., .eta., .theta. and .mu., and the "atypical" isoforms .zeta. and .lambda.. These isozymes have distinct patterns of tissue and organ localization (see Nishizuka, FASEB J., 1995, 9, 484-496 for review) and may serve different physiological functions.
The role of the individual PKC members has been studied by overexpression of the genes and, more recently, using antisense oligonucleotides. Overexpression of PKC-.delta. has been shown to inhibit cell growth and increased levels are associated with increased tumor potential. For example, PKC-.delta. is the PKC isoform most represented in murine erythroleukemia (MEL) cells. Incorporation of partially purified PKC-.delta. protein into permeabilized MEL cells causes a delay in chemically induced differentiation. Thus, it is believed the PKC-.delta. levels may be important in modulating differentiation in these leukemic cells (Fessino et al., Biochem J., 1995, 312, 549-554). However, growth effects may be dependent upon cell type. Modulation of PKC-.delta. may be particularly useful in hyperproliferative disorders, particularly hematopoietic diseases, such as acute promyelocytic, leukemia, and skin disorders, such as psoriasis.
According to the present invention, PKC-.delta. is also able to modulate tumor necrosis factor .alpha. expression. Modulation of PKC-.delta. may, therefore, also be useful in disease states associated with overexpression of TNF-.alpha., particularly infectious, inflammatory and autoimmune diseases. High levels of plasma TNF-.alpha. have been found in infectious diseases such as sepsis syndrome, bacterial meningitis, cerebral malaria, and AIDS; autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease (including Crohn's disease), sarcoidosis, multiple sclerosis, Kawasaki syndrome, graft-versus-host disease and transplant (allograft) rejection; organ failure conditions such as adult respiratory distress syndrome, congestive heart failure, acute liver failure and myocardial infarction (Eigler, A., et al., Immunol. Today, 1997, 18, 487-492). Other diseases in which TNF-.alpha. is involved include asthma (Shah, A., et al., Clinical and Experimental Allergy, 1995, 25, 1038-1044), brain injury following ischemia (Arvin, B., et al., Ann. NY Acad. Sci., 1995, 765, 62-71), non-insulin-dependent diabetes mellitus (Hotamisligil, G. S., et al., Science, 1993, 259, 87-90), insulin-dependent diabetes mellitus (Yang, X. -D., et al., J. Exp. Med., 1994, 180, 995-1004), hepatitis (Ksontini, R., et al., J. Immunol., 1998, 160, 4082-4089), atopic dermatitis (Sumimoto, S., et al., Arch. Dis. Child., 1992, 67, 277-279), and pancreatitis (Norman, J. G., et al., Surgery, 1996, 120, 515-521). Further, Suganuma, M., et al. (Cancer Res., 1996, 56, 3711-3715) suggest that inhibitors of TNF-.alpha. may be useful for cancer prevention. In addition, elevated TNF-.alpha. expression may play a role in obesity (Kern, P. A., J. Nutr., 1997, 127, 1917S-1922S). TNF-.alpha. was found to be expressed in human adipocytes and increased expression, in general, correlated with obesity.
Two major classes of drugs have been used to induce differentiation. Retinoic acids are used for the treatment of various leukemias (Chomienne, C., et al., FASEB J., 1996, 10, 1025-1030) and skin disorders (Orfanos, C. E., et al., Drugs, 1997, 53, 358-388). A major side effect of retinoic acids in their teratogenicity. Vitamin D3 derivatives are currently being studied for use in skin disorders (Gniadecki, R., Br. J. Pharmacol., 1997, 120, 1119-1127; Kobayashi, T., J. Dermatol. Sci., 1998, 16, 158-164).
Although numerous compounds have been identified as PKC inhibitors (see Hidaka and Hagiwara, Trends in Pharm. Sci., 1987, 8, 162-164 for review), few have been found which inhibit PKC specifically, much less specific isozymes of PKC. While the quinoline sulfonamide derivatives such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibit PKC at micromolar concentrations, they exhibit similar enzyme inhibition kinetics for PKC and the CAMP-dependent and cGMP-dependent protein kinases. Staurosporine, an alkaloid product of Streptomyces sp., and its analogs, are the most potent in vitro inhibitors of PKC identified to date. However, they exhibit only limited selectivity among different protein kinases (Gescher, Anti-Cancer Drug Design, 1989, 4, 93-105). Certain ceramides and sphingosine derivatives have been shown to have PKC inhibitory activity and to have promise for therapeutic uses, however, there remains a long-felt need for specific inhibitors of the enzymes.
There is also a desire to inhibit specific PKC isozymes, both as a research tool and in diagnosis and treatment of diseases which may be associated with particular isozymes. It is presently believed that different PKC isozymes may be involved in various disease processes depending on the organ or tissue in which they are expressed. Thus far, PKC isozyme specific antisense oligonucleotides have been used to study PKC-.alpha. (McGraw, K., et al., Anti-Cancer Drug Des., 1997, 12, 315-326), and an antisense oligonucleotide drug, ISIS 3521, targeted to PKC-.alpha. is presently in clinical trials and has demonstrated encouraging results in patients with solid tumours. Antisense oligonucleotides have also been used to inhibit PKC-.beta. (Gamard, C. J., Cell Growth Diff., 1994, 5, 405-409), PKC-.epsilon. (Traub, O., et al., J. Biol. Chem., 1997, 272, 31251-31257), and PKC-.zeta. (Liao, D. F., J. Biol. Chem., 1997, 272, 6146-6150). Diaz-Meco Conde et al. disclose a peptide corresponding to the pseudo-substrate region of PKC-.zeta. and oligonucleotides antisense to this isozyme (WO Application 93/20101). Alvaro et al. have identified a novel mutant form of PKC associated with tumors and disclose oligonucleotide sequences complementary to the mutant form (WO Application 94/29455).
Specific inhibitors of PKC-.delta. are believed to be useful for studying the precise role of this isozyme and for therapeutic applications. A compound isolated from Mallotus philippinensis, rotterlin, shows some specificity to PKC-.delta. relative to other PKC family members (Gschwendt, M., et al., Biochem. Biophys. Res. Commun., 1994, 199, 93-98). However, this compound also shows some inhibition of other protein kinases including calmodulin-dependent protein kinase III.
Antisense oligonucleotides specific for the PKC-.delta. isozyme have also been used. Liedtke, C. M., et al. (Am. J. Physiol., 1997, 273, C1632-C1640) used an oligonucleotide complementary to the translation initiation region of mouse PKC-.delta. to block .alpha..sub.1 -adrenergic activation of Na-K-2Cl cotransport. Pessino, A., et al. (Biochem. J., 1995, 312, 549-554) used an oligonucleotide complementary to the translation initiation region of PKC-.delta. to decrease PKC-.delta. levels and induce differentiation of murine erythroleukemia cells.
There are currently several approaches for directly inhibiting TNF-.alpha. expression. These include antibodies, human soluble TNF-.alpha. receptor (Camussi, G., Drugs, 1998, 55, 613-620) and oligonucleotides, including triplex-forming oligonucleotides, ribozymes, and antisense oligonucleotides. Examples of indirect TNF-.alpha. inhibitors include phosphodiesterase inhibitors (e.g. pentoxifylline) and metalloprotease inhibitors (Eigler, A., et al., Immunol. Today, 1997, 18, 487-492). Indirect inhibitors of TNF-.alpha., such as an inhibitor in the TNF-.alpha. signaling pathway, may provide means to inhibit a broad spectrum of activities associated with immune and inflammatory diseases.
There remains a long-felt need for improved compositions and methods for inhibiting PKC-.delta. gene expression.